Mutation Research/Genetic Toxicology最新文献

The aim of this study was to examine whether the storage of tissues at − 80°C or − 20°C affects the benzo[a]pyrene(B[a]P)-derived DNA adduct pattern and levels in rat tissues. Three rats were treated orally with a single dose of 100 mg B[a]P/kg b.w. and killed 24 h later. White blood cells (WBC) were isolated from the fresh blood. Livers, lungs and hearts were immediately removed, dissected into small fragments and were pooled for each organ. Pooled samples were proportionally divided into 7 aliquots. DNA from the first aliquot was immediately isolated (time 0). The other aliquots were frozen and stored at − 20°C or at − 80°C. DNA was isolated from the frozen samples at 1, 5 and 10 months later. ³²P-postlabeling analysis was performed at the beginning and at the end of study with the whole set of samples. Two B[a]P-derived adducts were detected in all tissues but with different intensities types. One of the adducts was found predominantly in WBC (∼ 85%) and liver (∼ 68%), while heart and lung accounted only for ∼ 43% and ∼ 39%, respectively. This adduct was tentatively identified as benzo[a]pyrene diol-epoxide-N² adduct (BPDE-N²-dG) based on TLC and HPLC analyses of ³²P-postlabeled adducts. The highest total DNA adduct level (sum of 2 spots) was found in lung (4.90 adducts/10⁸ nucleotides) compared with heart, liver and WBC (3.55, 2.37 and 2.32 adducts/10⁸ nucleotides, respectively). The analysis of variance provided evidence that storage of tissues at − 20°C or at −80°C up to 10 months did not significantly affect B[a]P DNA adduct levels and patterns in the rat lung, heart and liver. Our study indicates that properly stored tissues can be used for DNA adduct analysis with confidence.

1.8-Dihydroxyanthraquinones are under debate as plant-derived carcinogens that are found in laxatives, food colors, and possibly vegetables. Published genotoxicity data are controversial, and so three of them (emodin, danthron and aloe-emodin) were tested in a number of in vitro assay systems. All three compounds induced tk-mutations in mouse lymphoma L5178Y cells. Induction of micronuclei also occured in the same cell line, and was dose-dependent, with the potency ranking being danthron > aloe-emodin > emodin. In a DNA decatenation assay with a network of mitochondrial DNA of C. fasciulata, all three test compounds inhibited the topoisomerase II-mediated decatenation. Danthron and aloe-emodin, but not emodin, increased the fraction of DNA moving into comet tails when tested at concentrations around 50 μM in single-cell gel-electrophoresis assays (SCGE; comet assay). Comet assays were also used in modified form to determine whether pretreatment of the cells with the test compounds would reduce the effects of etoposide, a potent topoisomerase II inhibitor. All three test chemicals were effective in this pretreatment protocol, with danthron again being the most potent. Given clearcut evidence of their genotoxic activity, further research on the human cancer risk of these compounds may be warranted.

The main objective of this study was to compare the cytotoxic genotoxic and mutagenic activity of a number of chlorinated aliphatic hydrocarbons, which are widely used as chemical intermediates, solvents, degreasing agents etc. in industry, and to establish the structure-toxicity relationship of the chemicals by using the most adequate determinants in estimating their toxicity. The mutagenicity and cytotoxicity of some of the candidate chemicals, namely 1,2-dichloroethylene, 1,1,2-trichloroethane, 1,3-dichloropropane, 1,2,3-trichloropropane and 1,1,3-trichloropropene were evaluated in an in vitro micronucleus assay. The cytokinesis-block methodology was applied on human lymphocytes in the presence or absence of an external metabolic activation system (S9-mix). In the micronucleus assay, all test substances, except 1,2,3-trichloropropane with and without S9-mix and 1,1,2-trichloroethane without S9-mix in the repeated experiment, exhibited a low but statistically significant mutagenic activity, compared to the concurrent control. However, none of the five chemicals was able to induce a clear and reproducible linear dose-dependent increase in micronucleus frequencies in this assay. Generally, mutagenic activity of the chemicals was found in the absence of severe cytotoxicity and/or cell cycle delay. The DNA breakage capacity and the cytotoxicity of these chemicals were also assessed in the alkaline single cell gel (SCG) electrophoresis test (comet assay) with and without S9-mix in isolated human lymphocytes. All chemical compounds induced DNA breakage, in the presence or absence of the metabolic activation system, at the doses tested. The data showed that the DNA reactivity of the chemicals increased with increasing degree of halogenation. The results of the present work suggested that the comet assay might be a more suitable and sensitive screening method than the micronucleus test for this particular class of compound. However, both assays do detect different endpoints.

The effect of Tochu tea, which is an aqueous extract of Eucommia ulmoides leaves and a popular beverage in Japan, on the urine mutagenicity before and after ingestion of raw fish and cooked beef was studied using Salmonella typhimurium YG1024. Urines were collected from seven healthy, non-smoking Japanese women before and after ingestion of raw fish and cooked beef. In experiment 1, 3 were in a control group and 4 were in a Tochu tea-drinking group (2000 ml per day). The mutagenicity of urine from the Tochu tea-drinking group was much lower. In experiment 2 the 7 women switched groups; the tea-drinking group became the control group, and the control group became the Tochu tea-drinking group. Again, the mutagenicity of urine collected from the Tochu tea-drinking group was much lower. These results suggest that the decrease in the mutagenicity of the urine from the Tochu tea-drinking group was due to the intake of Tochu tea, but not to individual differences. Thus, the ingestion of Tochu tea may reduce human exposure to dietary mutagens.

Peripheral blood samples were collected from human volunteers at 0 (5–10 min before), and at 1 and 2 h after a single oral dose of 300 mg of melatonin. At each time point, (i) the concentration of melatonin in the serum and in the leukocytes were cultured with mitogenic stimulation to determine the extent of radiation-induced genetic damage, viz., chromosome aberrations and micronuclei. For each volunteer, the results showed a significant increase in the concentration of melatonin in the serum and in the leukocytes at 1 h after the oral dose of melatonin, as compared to the sample collected at 0 h. The lymphocytes in the blood samples collected at 1 and 2 h after melatonin ingestion and exposed in vitro to 150 cGy gamma radiation exhibited a significant decrease in the incidence of chromosome aberrations and micronuclei, as compared with similarly irradiated lymphocytes from the blood sample collected at 0 h; the frequencies abserved in the cells sampled at 2 h after the ingestion of melatonin were consistently lower when compared with those collected at 1 h. The data may have important implications for the protection of human lymphocytes from the genetic damage induced by free radical-producing mutagens and carcinogens.

The hypothesis that acrylamide induces dominant lethal mutations and heritable translocations in male mice, not through direct adduction, but by conversion to the reactive epoxide, glycidamide, was investigated. Three studies, namely, induction of dominant lethal mutations, heritable translocations, and unscheduled DNA synthesis in spermatids, which were conducted earlier in this laboratory for acrylamide, were also performed for glycidamide to determine its mutagenic properties and to compare responses. Results of these studies are consistent with the proposal that in vivo conversion to glycidamide is responsible for the mutagenicity of acrylamide in male mice.

The mutagenicity of 3 dihydroxybenzene (DHB) and 9 dihydroxynaphthalene (DHN) isomers was examined by using 5 different Ames Salmonella mutagenicity tester strains in the presence and absence of phenobarbital and 5,6-benzoflavonetreated rat liver S9-mix. Of the 3 DHB isomers, 1,4-DHB (hydroquinone) was mutagenic, and of the 9 DHN isomers, 1,3-DHN (naphthoresorcinol), 1,4-DHN (hydronaphthoquinone), 1,6-DHN and 1,7-DHN were mutagenic. Mutagenicity of all the compounds tested was observed in the absence of S9-mix, while 1,4-DHN and 1,6-DHN were also mutagenic in the presence of S9-mix. The mutagenicity of 1,4-DHB and 1,4-DHN for TA104, which is a strain sensitive to oxidative mutagens, was almost completely or partially inhibited by superoxide dismutase (SOD) and/or catalase, indicating the involvement of activated oxygen species in mutagenesis. Furthermore, from the finding that the 4 DHNs were mutagenic for TA2637, the strain sensitive to frameshift mutagens, it is possible that the mutagenicity of DHNs for S. typhimurium was also attributable to DNA adducts that form with quinones and/or semiquinones through oxidation of DHNs. The mutagenicity of 1,3-DHN, which showed the largest number of revertants in strains TA100, TA98, TA2637 and TA104, was greatly decreased, when their pKM101 plasmid-deficient strains, TA1535, TA1538, TA1537 and TA2659 were used. This observation suggests that an SOS repair system was involved in the mutagenesis of 1,3-DHN for S. typhimurium.

In this study the effect of piperonyl butoxide (PBO) on unscheduled DNA synthesis in precision-cut human liver slices has been examined. Liver slices prepared from tissue samples from five human donors were cultured in medium containing [³H]thymidine and 0–2.5 mM PBO using a dynamic organ culture system. After 24 h the liver slices were processed for autoradiographic examination of UDS. As positive controls, liver slices were also cultured with three known genotoxic agents, namely 2-acetylaminofluorene (2-AAF), aflatoxin B₁ (AFB₁) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). UDS was quantified as the net grain count in centrilobular hepatocytes and as the percentage of centrilobular hepatocyte nuclei with >5 and >10 net grains. Compared to control liver slice cultures PBO had no effect on UDS. In contrast, treatment with 0.02 and 0.05 mM 2-AAF, 0.002 and 0.02 and 0.02 mM AFB₁ and 0.005 and 0.05 mM PhIP produced significant increases in net grain counts of centrilobular hepatocytes. The greatest induction of UDS was observed in liver slices treated with 0.05 mM PhIP. Treatment with 2-AAF, AFB₁ and PhIP also produced increases in the number of centrilobular hepatocyte nuclei with >5 and >10 net grains. At the concentrations examined neither PBO, 2-AAF nor PhIP had any significant effect on replicative DNA synthesis in 24 h cultured human liver slices. In cultured liver slices treated with 0.02, but not 0.002, mM AFB₁ a significant reduction in the rate of replicative DNA synthesis was observed. These results demonstrate that PBO does not induce UDS in cultured human liver slices. However, all three positive control compounds produced marked significant increases in UDS, thus confirming the functional viability of the human liver slice

Phenethyl isothiocyanate (PEITC), a constituent of many cruciferous vegetables, is an effective chemopreventive agent against N-nitrosamine-induced carcinogenesis. We have investigated the extent to which PEITC modulates the clastogenicity of standard genotoxicants, mitomycin C and cyclophosphamide, using bone marrow cells of Swiss albino mice. PEITC, 1 μmol/kg body weight in corn oil was administered by gavage for 7 consecutive days to prime the animals. 24 h later, mice received a single dose of cyclophosphamide (10 or 20 mg/kg body weight)or mitomycin C (1 or 2 mg/kg body weight) intraperitoneally. Clastogenicity of the chemicals was compared using PEITC-primed and non-primed animals 24 h after clastogen treatment. As a single agent, PEITC is not clastogenic even after 7 days of priming. Oral priming with PEITC decreased the aberrations per cell values by 22–67% in all cases. PEITC could only alleviate the clastogenicity of I mg/kg body weight mitomycin C to near-control values (p ≤ 0.05). Although PEITC is reported to be effective against N-nitrosamine-induced tumorigenesis by preventing metabolic activation and by blocking the reactive species formed, it is virtually ineffective against the clastogenicity of cyclophosphamide. The results of inhibition by PIETC of the clastogenicity of mitomycin C suggest that the modulation of mitomycin C bio-activation contributes to, but may not be sufficient for, PIETC chemoprevention of clastogenicity by mitomycin C.

Some mixed disulfides derived from disulfiram and endogenous thiol compounds have been synthesized, biochemically characterized and their potential antigenotoxic effects have been proposed. The present study evaluated the mutagenic and antimutagenic specificities of two mixed disulfides using S. typhimurium reversion assay, namely S-(N,N-diethyldithiocarbamoly)-N acetylcysteine (AC-DDTC) and -L-glutathione (GS-DDTC). The two mixed disulfides were not mutagenic to Salmonella strains TA98 and TA100 in the presence or absence of S9 mixture. The increased number of revertants by benzo[a]pyrene (B(a)P) has been reduced to the control level by the preincubation with AC-DDTC or GS-DDTC. It was not due to the killing effect of B(a)P, mixed disulfides or B(a)P-disulfide mixture. The antimutagenic effect of AC-DDTC was more potent than that of GS-DDTC. These results indicate that AC-DDTC and GS-DDTC may have a role to play in reducing the risk of mutagenic effects of B(a)P.